Smash and Grab
- Grow cells in 3mls selective media o/n.
- Pellet cells by 2 quick spins in a microfuge.
- Resuspend cells in 200ul of the following soln:
- 5ml SCE
- 60ul 10mg/ml 100T Zymolyase
- 10ul 2-ME
- Vortex and inc at 37¡C for 30-60 min.
- Add 400ul 0.2N NaOH/1%SDS, invert to mix and inc. on ice for 5 min.
- Add 300ul 3M K/5M OAc pH4.5, invert to mix and inc on ice x 5 min.
- Spin 2 min in microfuge, pour super into a fresh tube and repeat spin. Remove 500ul to a fresh tube.
- Add 300ul iPrOH, vortex and let stand for 5 min. at RT.
- Pellet DNA 5 min in a microfuge, then wash pellet with 70% EtOH and repeat spin.
- Dry pellet in speed vac and recon in 25ul TE.
- Porate 2ul into electrocompetent coli, recover and plate all.
- 1M Sorbitol
- 0.1M Sodium Citrate pH 7.6
- 0.06M EDTA
Site designed by: David C. Amberg
Site maintained by: Blaine Bettinger
Department of Biochemistry and Molecular Biology
Last updated: November 2, 2001
All contents copyright© 2000
SUNY Upstate Medical University
750 East Adams Street
Syracuse, NY 13210